PROJECT SUMMARY Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults in the US. Proliferative DR (PDR) is an advanced stage of DR associated with the growth of abnormal retinal blood vessels, often referred to as neovascularization (NV). These neovessels can lead to hemorrhaging, vitreous scarring and retinal detachment, often resulting in irreversible vision loss. Numerous studies have shown that vascular endothelial growth factor (VEGF) plays an important role in the development of retinal NV associated with PDR. Laser treatment and/or anti-VEGF therapies are often used to control PDR; unfortunately, both have their drawbacks. Laser treatment is not curative and can damage healthy tissues. Anti-VEGF therapies, administered by intravitreal injection, carry the risk of endophthalmitis and are not efficacious in a significant fraction of diabetics. Methods allowing the informed modification of therapeutic regimes over the course of PDR are needed to improve patient outcomes. To develop such methods, retinal mRNAs associated with NV must be evaluated as predictive biomarkers and then exploited as a basis for retinal imaging. Retinal mRNA biomarkers offer the potential to image the onset, development and resolution of retinal NV in relation to therapeutic response. Interestingly, increased levels of endoglin mRNA occur in PDR. In experimental proliferative retinopathy, endoglin mRNA is detected at loci of incipient NV lesions and remains localized to these developing structures. Furthermore, anti-endoglin antibodies block experimental retinal NV. Taken together, these studies indicate that endoglin mRNA is intrinsically linked to NV development and progression suggesting that it is well-suited as a molecular target for imaging retinal NV. This proposal outlines a research plan to investigate the application of a novel strategy for transfection-free targeted delivery of short hairpin-RNA-lipid conjugates, incorporating an anti- sense sequence complimentary to endoglin (AS-ENG-shRNA-lipid). These AS-ENG-shRNA-lipid conjugates are rapidly internalized by cells and tissues, and they incorporate a dye that becomes fluorescently active upon hybridization to endoglin mRNA allowing their use as optical imaging probes. In this proposal, we describe experiments designed to test the capacity of AS-ENG-shRNA-lipid conjugates to image endoglin mRNA in retinal microvascular endothelial cells (RMEC) cultured under DR-relevant conditions. The mechanism(s) of AS-ENG- shRNA-lipid uptake by RMEC will also be determined. In vivo retinal imaging experiments will be performed in a mouse model of oxygen-induced retinopathy (OIR). OIR mice demonstrate a robust retinal neovascular response that is responsive to anti-VEGF therapy. We will test the capacity of AS-ENG-shRNA-lipid conjugates to longitudinally image retinal NV over the course of mouse OIR and in response to anti-VEGF therapy. Lastly, we will assess the in vivo biodistribution, pharmacokinetic, and toxicity of AS-ENG-shRNA-lipid conjugates in mice. This research will facilitate the clinical translation of this new imaging methodology for improved management of diabetic retinopathy and other ocular neovascular conditions. !